Conjugates of a glycoprotein or a glycan with a toxic payload

Abstract

The invention relates to a glycoprotein-toxic payload molecule conjugate, a toxic payload molecule-glycan conjugate, and a pharmaceutical composition. The invention further relates to a method for preparing the glycoprotein-toxic payload molecule conjugate, the method for modulating growth of a cell population and a method of treating tumour cells.

1 . A glycoprotein-toxic payload molecule conjugate represented by formula I
   [D-L-G] n -Gp  Formula I
   wherein   Gp is a glycoprotein comprising an N-glycan, wherein the N-glycan comprises a GlcNAc residue bound by a β-N linkage to an asparagine;   n is an integer from 1 to about 20;   D is a toxic payload molecule;   L is a linker group covalently joining G to D; and   G is a saccharide structure represented by formula II   
       
         
           
           
               
               
           
         
         wherein 
         R is a glycosidic bond to the N-glycan or a glycosidic bond to the GlcNAc residue bound by a β-N linkage to an asparagine; 
         X 1  is H or carboxyl; 
         X 2 , X 3  and X 4  are each independently OH, H, amino, C 2 -C 6  acylamide, phosphate or sulphate ester, or a bond to L; 
         X 5  is CH 2 OH, carboxyl, CH 3 , H, C 1 -C 3  alkyl or substituted C 1 -C 3  alkyl, or a bond to L; 
         with the proviso that one substituent selected from X 2 , X 3 , X 4  and X 5  is a bond to L or bonded via a bond to L; and 
         with the proviso that when X 1  is carboxyl, then X 2  is H, X 3  is OH, X 5  is C 1 -C 3  alkyl or substituted C 1 -C 3  alkyl; R is a glycosidic bond to the N-glycan; and X 4  is a bond to L or X 5  is bonded via a bond to L; or 
         when X 1  is H, then R is a glycosidic bond to the N-glycan or to the GlcNAc residue bound by a β-N linkage to an asparagine. 
       
     
     
         2 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein G is a saccharide structure represented by formula III 
       
         
           
           
               
               
           
         
         wherein 
         R is a glycosidic bond to the N-glycan; 
         X 4  is OH, H, amino, C 2 -C 6  acylamide, phosphate or sulphate ester, or a bond to L; 
         X 5  is C 1 -C 3  alkyl or substituted C 1 -C 3  alkyl; 
         and X 4  is a bond to L or X 5  is bonded via a bond to L. 
       
     
     
         3 . The glycoprotein-toxic payload molecule conjugate according to  claim 2 , wherein the N-glycan comprises a terminal Galβ residue and R is a glycosidic bond to the terminal Galβ residue. 
     
     
         4 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein the N-glycan consists of the structure represented by formula IV 
       
         
           
           
               
               
           
         
         wherein (β-N-Asn) is a β-N linkage to an asparagine and y is 0 or 1; 
         X 1  is H; 
         X 2 , X 3  and X 4  are each independently OH, H, amino, C 2 -C 6  acylamide, phosphate or sulphate ester, or a bond to L; 
         X 5  is CH 2 OH, carboxyl, CH 3 , H, C 1 -C 3  alkyl or substituted C 1 -C 3  alkyl, or a bond to L; 
         with the proviso that one substituent selected from X 2 , X 3 , X 4  and X 5  is a bond to L or bonded via a bond to L; and 
         R is a glycosidic bond to the GlcNAc residue. 
       
     
     
         5 . The glycoprotein-toxic payload molecule conjugate according to  claim 4 , wherein the anomeric structure of G is in β-D-galacto or β-D-gluco configuration and R is a glycosidic bond to the 4-position of the GlcNAc residue. 
     
     
         6 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein glycoprotein is capable of binding a target molecule. 
     
     
         7 . The glycoprotein-toxic payload molecule conjugate according to  claim 6 , wherein the glycoprotein-toxic payload molecule conjugate is internalised by a cell expressing the target molecule after the glycoprotein-toxic payload molecule conjugate is bound to the target molecule. 
     
     
         8 . The glycoprotein-toxic payload molecule conjugate according to  claim 6 , wherein the target molecule is selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD11, CD8, CD11a, CD19, CD20, CD22, CD25, CD26, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD46, CD52, CD56, CD79, CD105, CD138, epidermal growth factor receptor 1 (EGFR), epidermal growth factor receptor 2 (HER2/neu), HER3 or HER4 receptor, LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, EpCAM, alpha 4 /beta 7  integrin, alpha v/beta3 integrin including either alpha or beta subunits thereof, tissue factor (TF), tumor necrosis factor alpha (TNF-α), human vascular endothelial growth factor (VEGF), glycoprotein IIb/IIIa, TGF-beta, alpha interferon (alpha-IFN), IL-8, IL-2 receptor, IgE, respiratory syncytial virus (RSV), HIV-1 envelope glycoprotein gp120, cancer-associated high-mannose type N-glycans, blood group antigen Apo2, death receptor, flk2/flt3 receptor, obesity (OB) receptor, mp1 receptor, CTLA-4, transferrin receptor and protein C. 
     
     
         9 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein the glycoprotein is an antibody or a fragment thereof. 
     
     
         10 . (canceled) 
     
     
         11 . The glycoprotein-toxic payload molecule conjugate according to  claim 9 , wherein the glycoprotein is the antibody bevacizumab, tositumomab, etanercept, trastuzumab, adalimumab, alemtuzumab, gemtuzumab ozogamicin, efalizumab, rituximab, infliximab, abciximab, basiliximab, palivizumab, omalizumab, daclizumab, cetuximab, panitumumab, epratuzumab, 2G12, lintuzumab, nimotuzumab, GCM011, GCM012, or ibritumomab tiuxetan, or their glycoform antibody wherein the glycoform antibody comprises one or more introduced N-glycosylation sites in the light and/or heavy chain. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein L is a linker group represented by formula VIII 
       
         
           
           
               
               
           
         
         wherein 
         Y is an oxygen, sulphur, amine, amide, peptide or absent, wherein the peptide is an E 1 -P-E 2  unit in which E 1  and E 2  are independently C═O, O or NR p , wherein R p  is H, C 1 -C 6  alkyl or substituted C 1 -C 6  alkyl, P is a peptide unit from 2 to 5 amino acids in length, and E 1  and E 2  can independently be linked to the peptide through the terminal nitrogen, terminal carbon or through a side chain of one of the amino acids of the peptide; 
         Z is a saccharide or absent; 
         D′ is the toxic payload molecule, wherein the toxic payload molecule comprises an amine moiety, through which the toxic payload molecule is bound so as to form a secondary or tertiary amine; 
         R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8  and R 9  are each independently H, OH, amine, C 2 -C 6  acylamide, carboxyl, substituted carboxyl, C 1 -C 6  alkyl or substituted C 1 -C 6  alkyl; 
         W is H, CH 2 OH, CH 3 , carboxyl, substituted carboxyl, C 1 -C 6  alkyl or substituted C 1 -C 6  alkyl; 
         a is an integer from 0 to 6; 
         b is 0 or 1; 
         c and e are each independently an integer from 0 to 7; 
         d is an integer from 1 to 7; 
         Q is E′-F′-E, wherein F′ is an amine, amide, disulfide, thioether, thioester, hydrazone, Schiff base, oxime, olefin metathesis reaction product, triazole or phosphine group, or other group generated by the reaction of the functional group F-E and the functional group F′, wherein F is a functional group that can react with an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine, and F′ is an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine; and E is absent or a polyethyleneoxy unit of formula (CH 2 CH 2 O) p , wherein p is an integer from 2 to about 20; and E and E′ are each independently absent or a polyethyleneoxy unit of formula (CH 2 CH 2 O) p , wherein p is an integer from 2 to about 20; and 
         Q is bound via a bond to G. 
       
     
     
         16 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein L is a linker group represented by formula IX 
       
         
           
           
               
               
           
         
         wherein 
         Y is an oxygen, sulphur, amine, amide, peptide or absent, wherein the peptide is an E 1 -P-E 2  unit in which E 1  and E 2  are independently C═O, O or NR p , wherein R p  is H, C 1 -C 6  alkyl or substituted C 1 -C 6  alkyl, P is a peptide unit from 2 to 5 amino acids in length, and E 1  and E 2  can independently be linked to the peptide through the terminal nitrogen, terminal carbon or through a side chain of one of the amino acids of the peptide; 
         Z is a saccharide or absent; 
         D′ is the toxic payload molecule, wherein the toxic payload molecule comprises an amine moiety, through which the toxic payload molecule is bound so as to form a secondary or tertiary amine; 
         R 1 , R 2 , R 9  and R 10  are each independently H, OH, amine, C 2 -C 6  acylamide, carboxyl, substituted carboxyl, C 1 -C 6  alkyl or substituted C 1 -C 6  alkyl; 
         a is an integer from 0 to 6; 
         e is an integer from 0 to 3; 
         d and f are integers from 0 to 4 with the proviso that their sum is from 1 to 4; 
         Q is E′-F′-E, wherein F′ is an amine, amide, disulfide, thioether, thioester, hydrazone, Schiff base, oxime, olefin metathesis reaction product, triazole or phosphine group, or other group generated by the reaction of the functional group F-E and the functional group F′, wherein F is a functional group that can react with an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine, and F′ is an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine; and E is absent or a polyethyleneoxy unit of formula (CH 2 CH 2 O) p , wherein p is an integer from 2 to about 20; and E and E′ are each independently absent or a polyethyleneoxy unit of formula (CH 2 CH 2 O) p , wherein p is an integer from 2 to about 20; and 
         Q is bound via a bond to G. 
       
     
     
         17 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein L is a linker group represented by formula X 
       
         
           
           
               
               
           
         
         wherein 
         Y is an oxygen, sulphur, amine, amide, peptide or absent, wherein the peptide is an E 1 -P-E 2  unit in which E 1  and E 2  are independently C═O, O or NR p , wherein R p  is H, C 1 -C 6  alkyl or substituted C 1 -C 6  alkyl, P is a peptide unit from 2 to 5 amino acids in length, and E 1  and E 2  can independently be linked to the peptide through the terminal nitrogen, terminal carbon or through a side chain of one of the amino acids of the peptide; 
         Z is a saccharide or absent; 
         D′ is the toxic payload molecule, wherein the toxic payload molecule comprises an amine moiety, through which the toxic payload molecule is bound so as to form a secondary or tertiary amine; 
         R 1  and R 2  are each independently H, OH, amine, C 2 -C 6  acylamide, carboxyl, substituted carboxyl, C 1 -C 6  alkyl or substituted C 1 -C 6  alkyl; 
         a is an integer from 0 to 6; 
         c and e are each independently an integer from 0 to 3; 
         Q is E′-F′-E, wherein F′ is an amine, amide, disulfide, thioether, thioester, hydrazone, Schiff base, oxime, olefin metathesis reaction product, triazole or phosphine group, or other group generated by the reaction of the functional group F-E and the functional group F′, wherein F is a functional group that can react with an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine, and F′ is an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine; and E is absent or a polyethyleneoxy unit of formula (CH 2 CH 2 O) p , wherein p is an integer from 2 to about 20; and E and E′ are each independently absent or a polyethyleneoxy unit of formula (CH 2 CH 2 O) p , wherein p is an integer from 2 to about 20; and 
         Q is bound via a bond to G. 
       
     
     
         18 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein D-L-G is selected from the group consisting of D-aminooxyacetic acid-7-aldehydo-NeuAc, D-aminooxyacetic acid-7-aldehydo-NeuAc, N-(6-N 3 -Gal)-D-(triazole)-ABAA-sialic acid oxime, N-(6-N 3 -Gal)-D-(triazole)-ABAA-sialic acid oxime, triazole conjugate of 9-azido-NeuAc and N-(6-O-propargyl-D-galactosyl)-D, ABAA-D-7-aldehydo-NeuNAc, D-TREA-DBCO-9-azido-NeuNAc, D-TRSLac-Lys-DBCO-9-azido-NeuNAc, D-DBCO-9-azido-NeuNAc, D-Val-Cit-PAB-DBCO-9-azido-NeuAc, conjugate of N-(6-O-propargyl-D-galactosyl)-D and 9-azido-NeuAc, conjugate of N-(6-O-propargyl-D-galactosyl)-D and 9-azido-NeuAc, conjugate of N-(6-O-propargyl-D-galactosyl)-D and 9-azido-NeuAc, conjugate of N-(6-O-propargyl-D-galactosyl)-D and 9-azido-NeuAc, and conjugate of N-(6-O-propargyl-D-galactosyl)-D and 9-azido-NeuAc, wherein D is a toxic payload molecule, or wherein D-L-G is selected from the group consisting of monomethyldolastatin-aminooxyacetic acid-7-aldehydo-NeuAc, monomethylauristatin-aminooxyacetic acid-7-aldehydo-NeuAc, N-(6-N 3 -Gal)-MODO-(triazole)-ABAA-sialic acid oxime, N-(6-N 3 -Gal)-MODO-(triazole)-ABAA-sialic acid oxime, triazole conjugate of 9-azido-NeuAc and N-(6-O-propargyl-D-galactosyl)-monomethyldolastatin 10, ABAA-MODO-7-aldehydo-NeuNAc, MODO-TREA-DBCO-9-azido-NeuNAc, MODO-TRSLac-Lys-DBCO-9-azido-NeuNAc, DM1-DBCO-9-azido-NeuNAc, MODO-Val-Cit-PAB-DBCO-9-azido-NeuAc, conjugate of N-(6-O-propargyl-D-galactosyl)-epirubicin and 9-azido-NeuAc, conjugate of N-(6-O-propargyl-D-galactosyl)-doxorubicin and 9-azido-NeuAc, conjugate of N-(6-O-propargyl-D-galactosyl)-daunorubicin and 9-azido-NeuAc, conjugate of N-(6-O-propargyl-D-galactosyl)duocarmycin MA and 9-azido-NeuAc, and conjugate of N-(6-O-propargyl-D-galactosyl)duocarmycin and 9-azido-NeuAc. 
     
     
         19 . (canceled) 
     
     
         20 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein the toxic payload molecule is a dolastatin, auristatin, doxorubicin, DM1, epirubicin, duocarmycin, or any analogue or derivative thereof. 
     
     
         21 . The glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein the glycoprotein-toxic payload molecule conjugate is selected from the group consisting of monomethyldolastatin-aminooxyacetic acid-cetuximab conjugate, monomethylauristatin-aminooxyacetic acid-cetuximab conjugate, monomethyldolastatin-aminooxyacetic acid-levulinyl-cetuximab conjugate, N-(6-N 3 -Gal)-MODO-(triazole)-ABAA-sialic acid oxime-cetuximab conjugate, N-(6-N 3 -Gal)-MODO-(triazole)-ABAA-sialic acid oxime-Endo S-treated cetuximab conjugate, triazole conjugate of 9-azido-NeuAc-cetuximab and N-(6-O-propargyl-D-galactosyl)-monomethyldolastatin 10, ABAA-MODO-7-aldehydo-NeuNAc-trastuzumab conjugate, ABAA-MODO-7-aldehydo-NeuNAc-anti-CD33 conjugate, ABAA-MODO-7-aldehydo-NeuNAc-afucosyl trastuzumab conjugate, MODO-TREA-DBCO-9-azido-NeuNAc-G2F-trastuzumab conjugate, MODO-TRSLac-Lys-DBCO-9-azido-NeuNAc-G2F-trastuzumab conjugate, DM1-DBCO-9-azido-NeuNAc-G2F-cetuximab conjugate, MODO-Val-Cit-PAB-DBCO-9-azido-NeuAc-cetuximab conjugate, conjugate of N-(6-O-propargyl-D-galactosyl)-epirubicin and 9-azido-NeuAc-cetuximab, conjugate of N-(6-O-propargyl-D-galactosyl)-doxorubicin and 9-azido-NeuAc-cetuximab, conjugate of N-(6-O-propargyl-D-galactosyl)-daunorubicin and 9-azido-NeuAc-cetuximab, conjugate of N-(6-O-propargyl-D-galactosyl)duocarmycin MA and 9-azido-NeuAc-cetuximab, conjugate of N-(6-O-propargyl-D-galactosyl)duocarmycin and 9-azido-NeuAc-cetuximab, ABAA-MODO-7-aldehydo-NeuNAc-cetuximab and ABAA-MODO-7-aldehydo-NeuNAc-GMC012. 
     
     
         22 . (canceled) 
     
     
         23 . A method for preparing a glycoprotein-toxic payload molecule conjugate according to  claim 1 , wherein the method comprises the steps of:
 providing a glycoprotein comprising an N-glycan comprising an acceptor site; and   reacting a donor molecule with the glycoprotein comprising an N-glycan comprising an acceptor site in the presence of a glycosyltransferase;   wherein the donor molecule is represented by formula XI
   L′-G  Formula XI
 
   wherein G is a saccharide structure represented by formula XII   
       
         
           
           
               
               
           
         
         wherein 
         R is CMP, UDP or GDP; 
         X 1  is H or carboxyl; 
         X 2 , X 3  and X 4  are each independently OH, H, amino, C 2 -C 6  acylamide, phosphate or sulphate ester, or a bond to L′; 
         X 5  is CH 2 OH, carboxyl, CH 3 , H, C 1 -C 3  alkyl or substituted C 1 -C 3  alkyl, or a bond to L′; 
         with the proviso that one substituent selected from X 2 , X 3 , X 4  and X 5  is a bond to L′ or bonded via a bond to L′; 
         with the proviso that when X 1  is carboxyl, then X 2  is H, X 3  is OH, X 5  is C 1 -C 3  alkyl or substituted C 1 -C 3  alkyl; R is CMP; and X 4  is a bond to L′ or X 5  is bonded via a bond to L′; or 
         when X 1  is H, then R is UDP or GDP; 
         and wherein 
         L′ is D-L, wherein D is a toxic payload molecule and L is a linker group covalently joining G to D, or L′ comprises F-E, wherein F is a functional group that can react with an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine, and E is absent or a polyethyleneoxy unit of formula (CH 2 CH 2 O) p , wherein p is an integer from 2 to about 20. 
       
     
     
         24 .- 39 . (canceled) 
     
     
         40 . A method for modulating growth of a cell population expressing a target molecule, wherein the method comprises the step of
 contacting the glycoprotein-toxic payload molecule conjugate according to  claim 1  with the cell population, and optionally, wherein the cell population is a cancer cell population.   
     
     
         41 . The method according to  claim 40 , wherein the target molecule is a target molecule selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD11, CD8, CD11a, CD19, CD20, CD22, CD25, CD26, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD46, CD52, CD56, CD79, CD105, CD138, epidermal growth factor receptor 1 (EGFR), epidermal growth factor receptor 2 (HER2/neu), HER3 or HER4 receptor, LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, EpCAM, alpha 4 /beta 7  integrin, alpha v/beta3 integrin including either alpha or beta subunits thereof, tissue factor (TF), tumor necrosis factor alpha (TNF-α), human vascular endothelial growth factor (VEGF), glycoprotein IIb/IIIa, TGF-beta, alpha interferon (alpha-IFN), IL-8, IL-2 receptor, IgE, respiratory syncytial virus (RSV), HIV-1 envelope glycoprotein gp120, cancer-associated high-mannose type N-glycans, blood group antigen Apo2, death receptor, flk2/flt3 receptor, obesity (OB) receptor, mp1 receptor, CTLA-4, transferrin receptor and protein C. 
     
     
         42 .- 44 . (canceled)